Method for determining glucose levels in blood and other biological fluids



Nov. 2l, 1961 L. A. DoBRlcK 3,009,862 METHOD FOR OETERMINING GLucOsELEvELs IN BLOOD AND OTHER BIOLOGICAL FLUIDS Filed Oct. 6, 1958 (300,280)I--EXPERIn-:NTAL

v (20o |20) H--STOICHIOMETRIC I l I 200 400 600 800 |000 MICROGRAMSGLUCOSE Sttes Unit@ This invention relates generally to a rapid methodfor determining glucose levels in blood and other biological fluids andmore speciically to a rapid method for screening for blood glucose,which method is applied directly to protein-carrying blood serum orplasma.

Heretofore the usual method for determining glucose in biological fluidssuch as blood has required the preliminary steps (l) obtaining aprotein-free filtrate by precipitating the protein with such reagents assulfuric acid and sodium tungstate, or barium hydroxide and zincsulfate, and (2) removing the precipitated protein before proceeding todetermine the glucose content by the usual method employing coppersulfate or potassium ferricyanide. The requirement for a protein-freefiltrate makes such methods time consuming and generally unsuitable formass screening for glucose.

Another method heretofore used to determine glucose in biological uidssuch as blood serum employs the property of the enzyme, glucose oxidase,to catalyze the oxidation of glucose to gluconic acid. The amount ofglucose present in the blood serum is determined by manometricallymeasuring the oxygen uptake during the reaction. However, this techniqueis too complex for general use and therefore is totally unsuitable formass screening of large numbers of blood samples.

Another method heretofore used employs a paper tape which because of thehydrogen peroxide liberated by the glucose-glucose oxidase reactionyields a chromogenic oxidation product, the color intensity of which issemiquantitatively proportional to glucose concentration in urine andblood plasma. However, this method is not sufficiently quantitativelyaccurate, particularly when applied to blood.

It is therefore an object of this invention to provide an improvedmethod for determining glucose levels directly in biological fluids suchas blood serum or plasma which method does not require removal ofprotein from the serum or plasma for determination of glucose.

Another object of this invention is to provide an improved method forrapidly screening for glucose in biological fluids such as blood serumor plasma which method does not require comparison of the reactant witha standard, or comparison of the color of the reactant with a colorchart.

Another object of this invention is to provide an improved method formass screening for glucose in biological iiuids such as blood serum orplasma which is simple to perform, does not require special laboratoryskills or techniques, has a high degree of accuracy, and which overcomesthe disadvantages of the prior art methods.

Another object of the invention is to provide in a glucose-glucoseoxidase reaction a hydrogen acceptor which will itself indicate thecourse of the reaction.

Still another object of this invention is to provide an improved methodfor mass screening for glucose in biological iluids such as blood serumwhich method may be performed under either anaerobic or aerobicconditions.

Other objects and advantages will become readily apparent from thefollowing description of the invention taken together with theaccompanying drawing.

The inventor hereof found upon bringing together a jatt biological uidsuch as blood serum or plasma, an oxidation-reduction indicator, such assodium 2,6-dichlorobenzenoneindophenol, and a catalytic enzyme, such asglucose oxidase, in the foregoing order, that he obtained a reactionwhich if buffered at a pH of from about 4.0 to 5.6, with a preferred pHof 4.6, gave a solution varying from colorless to a deep blue. Thepresence or absence of color was found to depend on the quantities ofglucose, indicator, and enzyme present, and the pH of the solution. lfthe quantities of indicator and enzyme, the value of pH, and thereaction time are iixed within predetermined limits so as to establish ascreening level for glucose of a predetermined amount, then a colorlesssolution indicates glucose above this amount and a colored solutionglucose below this amount.

If the foregoing method is performed under anaerobic conditions, it ispossible to determine glucose concentrations Varying as little as l0milligrams per 10,0 milliliters above or below a set screening level.Thus glucose in a biological Huid such as blood serum or plasma can beclassified as being greater or less than any set screening level with anaccuracy 10 milligrams per 100 milliliters.

Anaerobic conditions may be obtained without the use of specialapparatus by the addition of a light oil, such as a light petroleum oil,to the blood serum or plasma before adding the indicator and enzyme.Under aerobic conditions the foregoing method also gives consistentresults with a probable error of about plus or minus 20 milligrams permilliliters.

The method of this invention owes its efficacy to the fact that theoxidation-reduction indicator, sodium 2,6- dichlorobenzenone-indophenolreplaces molecular oxygen as the hydrogen acceptor in theglucose-glucose oxidase reaction. Under anaerobic conditions, it hasbeen established experimentally that a sharply defined quantitativerelationship exists between the amount of glucose oxidized and theamount of sodium 2,6dichlorobenzenone-indophenol reduced. It has alsobeen confirmed experimentally that for every increment of l0() parts ofglucose oxidized, there is a corresponding increment of parts of sodium2,6-dichloroben- Zenone-indophenol (hereinafter referred to asindophenol) reduced. Whereas l mole of glucose will theoretically reducel mole of indophenol, the incremental ratio be- 'tween glucose oxidizedand indophenol reduced approaches stoichiometric proportions.

In the drawing curve I shows maximal amount of indophenol having aformula weight of 340, as determined iodometrically, reduced tocolorless state with increasing amounts of glucose, as determinedexperimentally. Curve 1I shows the stoichiometric relationship betweenindoi phenol having a formula weight of 340, as determinediodometrically, reduced to colorless state with increasing amounts ofglucose.

' The use of indophenol as an indicator gives a convenient means offollowing the glucose-glucose oxidase reaction since the dye isintensely colored in its oxidized state and totally colorless in itsreduced state.

The indicator indophenol may be prepared as a stock solution or it maybe prepared in powdered forml by mixing with a iiller such as sodiumchloride. Such powdered mixture may be formed into a stable standardizedtablet, vial or capsule. In any of the foregoing forms the indicator isdiluted with a suitable dilution reagent comprising an aqueous solutioncontaining 40 percent :by volume of propylene glycol, l percent byvoltune of ethyl alcohol and 0.2 percent by weight of sodium bicarbonatebefore use in the method of the invention. When diluted to apredetermined amount as described -dium vhydrogen phosphate. -citricacid-1H2O and 16.6 grams of anhydrous disodium -phosphate are diluted to100 milliliters with water and hereinafter, the indicator is ready foruse as a screening indicator.

The quantity of indophenol which the screening indicator solution iscaused to contain will depend upon the glucose screening level chosen.I'f the glucose screenmg level selected is 100 milligrams per 100milliliters, the

Vindicator is diluted with the dilution reagent until the plasma (in thepresence of an appropriate catalytic enzyme), only serum or plasmacontaining glucose in excess of the screening level will exhibitcolorless reaction products.

For the stock indicator solution 100 milligrams of indophenol is dilutedto 100 milliliters with the dilution reagent. By further dilutingappropriate aliquots of this stock indicator solution with the dilutionreagent an indicator screening solution is prepared. Each milliliter ofindicator screening solution so prepared Will contain a predeterminednumber of micrograms of indophenol vwhich has a formula weight, 340`(sodium, 2,6-dichlorobenzenone-indophenel-7.78H2O as determinediodometrically).

An lalternate method of preparing the indicator screen- .ing solutiondirectly by weight is to utilize the powdered mixture of indophenol andsodium chloride. For example, for the preparation of a screeningindicator solution .at kthe 100 milligram per 100 milliliters glucoselevel,

3.7900 grams of sodium chloride are intimately mixed with 0.2100 gram ofindophenol to give a total weight of 4.0000 grams. Of the foregoingmixture, 0.4000 gram is diluted to 45 milliliters with the dilutionreagent to directly give the screening indicator solution.

If screening levels other than 100 milligrams per 100 milliliters aredesired, the indicator is diluted with the dilution reagent until thesolution contains 60 micrograms of indophenol in excess of that which isvisibly reduced to the colorless state by 0.5 milliliter of glucose ofthe desired standard. For example, in a screening indicator solutionprepared yfor a 120 milligrams per 100 milliliters glucose level, theamount of indophenol which is visibly reduced to colorless state is 840micrograms. The total amount of indophenol, therefore, in such ascreening solution is 900 micrograms.

The dilution reagent used todilute the indicator contains proplyleneglycol to insure that the indophenol dye in the presence of the enzymeand the glucose remains soluble at the pH of from about 4.0 to 5.6, andparticularly at the preferred pH of about 4.6. Ethylene glycol may besubstituted for the propylene glycol to perform the same function.

Ethyl alchol is included in the dilution reagent to retard in thescreening reaction reoxidation of the reduced indophenol by any hydrogenperoxide which may be present.

The enzyme used in the method of the invention may 4be prepared from astable stock solution or it may be prepared directly from the anhydrousform. The stock solution used contained substantially 750 units permilliliter of glucose oxidase. The glucose oxidase is butteredpreferably tothe pH of about 4.6, by the addition of any suitable:butter such as citric acid and anhydrous diso- For example, 14.0 gramsof mixed with slight Warming until dissolved to give a stock buffer of apH of about 4.6. To obtain a buffer-enzyme mixture f 75 units permilliliter, for example, 6 parts by volume of the stock buffer are mixedwith 3 parts of water and one part of glucose oxidase. `Otherproportions of glucose oxidase may be used to give bufferenzyme mixturesof higher enzyme concentrations.

With the yforegoing materials on hand, the method of this invention maybe performed for the mass screening of biological Ifluids such as bloodserum or plasma. The term biological iiuid as used herein means anyfluid containing living or dead plant or animal matter, and is notlimited to uid derived from man or other animals.

If mass screening for glucose at a level of milligrams per 10'()milliliters is desired, the method of the invention will be performed asfollows:

A plurality of biological duid samples such as blood serum orV plasmaspecimens are placed in respective test tubes so that each tube contains0.5 milliliter of a particular specimen. Prior to placing the specimenin the tube, each tube is equipped with a short length of glass rod. Toeach tube is further added in the following order, l milliliter of lightperoleum oil (if anaerobic conditions are desired), 1.5 milliliters ofscreening indicator solution, and 0.5 milliliter of a 75-unitbuiTer-enzyme mixture, all previously prepared as described.

The test tubes are agitated gently, as by placing them in a rack androtating the rack so as to insure mixing of the reagents. As the rack isrotated the glass rods rotate in the tubes and assist in the mixing. Thetubes are then placed in a water bath at preferably 37 to 40 degreescentigrade, although the reaction will proceed, though more slowly, atroom temperature, and are again gently agitated at fteen minuteintervals. The temperature 37 to 40 degrees centrigrade is the preferredtemperature because at this temperature the results attain the greatestdegree of accuracy.

After a reaction time, preferably of thirty minutes, but which may be aslittle as tive minutes if a bufferenzyme mixture having a concentrationgreater than 75 units per milliliter of glucose oxidase is used and ifthe glucose concentration in the specimen being tested is above thepredetermined limit, a point of delineation between colored andcolorless solutions is observable. The tubes are then scanned, as byvisual observation or by means of an electro-mechanical color scanningdevice of conventional type, for changes in color. A substantiallyunchanged deep blue indicates glucose levels below 100 milligrarns per100 milliliters. A light blue color indicates glucose levels approaching100 milligrams per 100 milliliters, whereas a colorless solutionindicates glucose levels above 100 milligrams per 100 milliliters.

Using the method of this invention, a single individual is at least ableto visually screen approximately 100 blood specimens in one hour. Withthe aid of automatic equipment to deliver the reagents and to scan theserum specimens, the number of specimens screened may be greatlylncreased.

Experimentally it has been shown that protein does not affect theover-all reaction between indophenol `and glucose. For that reason, noprotein correction is needed in the final results.

While indophenol of formula weight 340 as determined iodometrically hasbeen used in the examples described herein, indophenol of other puritymay be used for the indicator in the method of this invention withsimilar results.

Although in the examples described herein sodium2,6-dichlorobenzenone-indophenol was the indicator used, sodium2,6-dibromobenzenone-indophenol may be substituted for sodium2,-6-dichlorobezenone-indophenol in the method of this invention withequivalent results, provided proper allowance is made for the differencein formula Weight and purity of the two indophenols.

It will be apparent to one skilled in the art that various changes andmodifications, singly or collectively, including the use of equivalentreagents and materials, may be made in the method' of the inventionwithout departing fromthe scope thereof as defined in the appendedclaims and without departing from the essence of the invention.

I claim:

1. A self-indicating method for directly screening for glucose at apredetermined level in a biological liuid comprising the steps ofbringing together in solution a predetermined amount ofglucose-containing biological fluid, an amount of an oxidation-reductionindicator selected from the class consisting of sodium2,6-dichlorobezenone-indophenol and sodium2,6-dibromobezenoneindophenol, said amount of said indicator beingsuflicient to change color as it is reduced in response to levels ofglucose in said biological uid equal to and above said predeterminedlevel, and an amount of a catalytic enzyme consisting of glucose oxidasebuffered to a pH of from about 4.() to about 5.6 to prevent saidindicator from being reoxidized suiciently to interfere with said colorchange of said indicator at said predetermined glucose level, saidamount of said endyme being suicient to act as a catalyst in thereduction of said indicator by said glucose, and warming said solutionto a predetermined temperature for a period suiicient to reduce saidindicator.

2. A self-indicating method for directly screening for glucose at apredetermined level in a biological fluid comprising the steps ofbringing together in solution in the following order a predeterminedamount of glucosecontaining biological fluid, an amount of a lightpetroleum oil suicient to establish substantially anaerobic conditionsfor said solution an amount of an oxidation-reduction indicator selectedfrom the class consisting of sodium 2,6-dichlorobenzenone-indophenol andsodium 2,6dibromobenzenone-indophenol, said amount of said indicatorbeing suflicient to change color as it is reduced in response to levelsof glucose in said biological fluid equal to and above saidpredetermined level, and an amount of catalytic enzyme consisting ofglucose oxidase buffered to a pH in the range of from about 4:0 to 5.6,said pH range cooperating with said oil to prevent said indicator frombeing reoxidized sufciently to interfere with said color change at saidpredetermined level, said amount of said enzyme being suflicient to actas a catalyst in the reduction of said indicator by said glucose, andwarming said solution to a predetermined temperature for a periodsucient to reduce said indicator.

3. A self-indicating method for directly screening for glucose at apredetermined level in a biological fluid selected from the classconsisting of blood serum and plasma comprising the steps of bringingtogether in solution a predetermined amount of glucose-containingbiological uid selected from the class consisting of blood serum andplasma, an amount of an indicator consisting of sodium2,-dichlorobenzenone-indophenol suflicient as it acts to indicate whileacting as a hydrogen acceptor to indicate directly by color changelevels of glucose in said biological uid equal to and above saidpredetermined level, and an amount of a catalytic enzyme consisting ofglucose oxidase buffered to a pH of 4.6 to prevent said indicator frombeing reoxidized suiciently to interfere with said color change of saidindicator at said predetermined glucose level, said amount of saidenzyme being sufficient to act as a catalyst in the reduction of saidindophenol by said glucose, and warming said solution at a temperatureof from about 37 to about 40 degrees centigrade for a period suicient tocomplete said reduction of said indicator.

4. A self-indicating method for directly screening for glucose at apredetermined level in a biological fluid selected from the classconsisting of blood serum and plasma comprising the steps of bringingtogether in solution in the following order a predetermined amount ofglucose-containing biological `liuid selected from the class consistingof blood serum and plasma, an amount of a light petroleum oil suicientto establish substantially anaerobic conditions for said solution, anamount of sodium 2,6-dichlorobenzenone-indophenol sufficient whileacting as a hydrogen acceptor to indicate by changing color in responseto the glucose in said biological fluid levels of glucose equal to andabove said predetermined level, and an .amount of a catalytic enzymeconsisting of glucose oxidase buffered to a pH of 4.6 to prevent saidindicator from being reoxidized suiciently to interfere with said colorchange of said indicator at said predetermined glucose level, saidamount of said enzyme being suflicient to act as a catalyst in thereduction of said sodium 2,6-dichlorobenzenone-indophenol by saidglucose, and warming said solution at a temperature of from about 37 to-about 40 degrees centigrade for a period suflicient to reduce saidindicator.

5. A self-indicating method for directly screening for glucose -at apredetermined level in a biological lluid comprising the steps ofbringing together in solution in the following order a predeterminedamount of glucosecontaining biological fluid, an amount of lightpetroleum oil suicient to establish substantially anaerobic conditionsfor said solution, a quantity of an aqueous screening indicator reagentcontaining a predetermined quantity of sodium2,6-dichlorobenzenone-indophenol capable of changing color when it isreduced in response to levels of glucose in said biological fluid equalto and above said predetermined level, said predetermined quantityexceeding by about 60 micrograms the amount of said sodium2,6-dichlorobenzenone-indophenol required to oxidize an `amount ofglucose corresponding to said predetermined level, and an amount o-fglucose oxidase buffered to a p-H of 4.6, said pH cooperating with saidoil to prevent said indicator from being reoxidized suiciently tointerfere with said `color change of said indicator at saidpredetermined level, said amount of said glucose being suicient to actas a catalyst in the reduction of said indophenol, and warming saidsolution at a temperature of from about 37 to about 40 degreescentigrade for a period sufficient to reduce said indicator.

6. A self-indicating method for directly screening for glucose Vat apredetermined level in a biological fluid comprising the steps ofbringing together in solution a predetermined amountV ofglucose-containing biological fluid, a quantity of an aqueous screeningindicator reagent containing a predetermined quantity of sodium2,6-dichlorobenzenone-indophenol capable of changing color after it isreduced in response to levels of glucose in said biological fluid equalto and above said predetermined level, said predetermined quantityexceeding by about l60 micrograms the amount of said sodium2,6-dichlorobenzenone-indophenol required to theoretically oxidize anamount of glucose corresponding to said predetermined level, and anamount of glucose oxidase buffered to a pH of 4.6 to prevent saidindicator from being reoxidized suiciently to interfere with said colorchange at said predetermined level, said amount of said glucose oxidasebeing sufiicient to act as a catalyst in the reduction of saidindophenol, and warming said solution at a temperature of from about 37to about 40 degrees centigrade for a period suiiicient to reduce saidindicator.

7. A self-indicating method for directly screening for glucose at apredetermined level in a biological fluid selected from the classconsisting of blod serum and plasma comprising the steps of bringingtogether in solution in the following order one part by volume ofglucosecontaining biological uid selected from the class consisting ofblood serum and plasma, two parts by volume of light petroleum oil toprovide substantially anaerobic conditions for said solution, threeparts by volume of an aqueous screening indicator reagent containing apredetermined quantity of sodium 2,6-dichlorobenzenone-indophenolcapable of changing color when it is reduced in response to levels ofglucose in said biological iluid equal to and above said predeterminedlevel, said predetermined quantity exceeding by about 60 micrograms theamount of said indophenol required to oxidize an amount of glucosecorresponding to said predetermined level, and one part glucose oxidaseenzyme buiered to a pH of 4.6, said pH cooperating with said oil toprevent said indicator from being reoxidized sufliciently to interferewith said color change of said indicator at said predetermined level andwarming said solution at a temperature of from about 37 to about 40degrees centigrade for a period suicient to reduce said indicator.

8. A self-indicating method for directly screening for glucose at apredetermined level in a biological fluid selected from the classconsisting of blood serum and plasma comprising the steps of bringingtogether in solution one part by volume of glucose-containing biologicalfluid selected from the class consisting of blood serum and plasma,three parts by volume of an aqueous screening indicator reagentcontaining a predetermined quantity of sodium2,6-dichlorobenzenone-indophenol capable of changing color when it isreduced in response to levels of glucose Iin said biological fluid equalto and above said predetermined level, said predetermined quantityexceeding by about 60 micrograms the amount of said indophenol requiredto oxidize an amount of glucose corresponding to said predeterminedlevel, and one part of glucose oxidase enzyme buffered to a pH of 4.6 tosubstantially prevent reoxidation of said indophenol, and warming saidsolution at a temperature of from about 37 to 4() degrees centigrade fora period suicient to reduce said indicator.

9. A self-indicating method for directly screening for glucose at apredetermined level thereof in a biological uid comprising the steps ofbringing together in solution a predetermined amount ofglucose-containing biological uid, an amount of an oxidation-reductionindicator selected from the class consisting of sodium2,6-dichlorobezenone-indophenol and sodium2,6-dibromobenzenoneindophenol, said amount of said indicator beingsulicent to change color when it is reduced in response to levels ofglucose in said biological iluid equal to and above said predeterminedlevel, an amount of a solvent for said indicator selected from the classconsisting of propylene glycol and ethylene glycol, said amount of saidsolvent being sufficient to maintain the solubility of said indicator insaid solution at a pH range of from about 4.0 to about 5.6, and anamount of a catalytic enzyme consisting of glucose oxidase buffered to apH in the range of from about 4.0 to about 5.6 to prevent said indicatorfrom being reoxidized suiciently to interfere with said color change ofsaid indicator at said predetermined glucose level, said amount of saidenzyme being suiiicient to act as a catalyst in the reduction o f saidindicator by said glucose, and Warming said solution to a predeterminedtemperature for a period sufiicient to reduce said indicator.

l0. A self-indicating method for directly screening for glucose at apredetermined level thereof in a biological lluid comprising the stepsof bringing together in solution a predetermined amount of aglucose-containing biological fluid, an amount of a light petroleum oilsuflicient to establish substantially anaerobic conditions for saidsolution, an amount of an oxidation-reduction indicator selected fromthe class consisting of sodium 2,6-dichlorobenzenone-indophenol andsodium 2,6-dibromobenzenone-indophenol, said amount of said indicatorbein-g sufiicient to change color when it is reduced in response tolevels of glucose in said biological iluid equal to and above saidpredetermined level, an amount of a solvent for said indicator selectedfrom the class consisting of propylene glycol and ethylene glycol, saidamount of said solvent being suflicient to maintain the solubility ofsaid indicator in said solution at a pH range of from about 4.0` toabout 5.6, and an amount of catalytic enzyme consisting of glucoseoxidase buffered to a pH in the range of from about 4.0 to about 5.6,said pH range cooperating with said oil to prevent said indicator frombeing reoxidized sutiiciently to interfere with said color change atsaid predetermined glucose level, said amount of said enzyme beingsufcient to act as a catalyst in the reduction of said indicator by saidglucose, and warming said solution to a predetermined temperature for aperiod suicient to reduce said indicator.

ll. A self-indicating method for directly screening for glucose at apredetermined level thereof in a biological uid comprising the steps ofbringing together in solution under substantially anaerobic conditions apredetermined amount of glucose-containing biological fluid, an amountof an oxidation-reduction indicator selected from the class consistingof sodium 2,6dichlorobenzenone-indophenol and sodium2,6-dibromobenzenone-indophenol, said amount of said indicator beingsuicient to change color When it is reduced in response to levels ofglucose in said biological iluid equal to and above said predeterminedlevel, an amount of a solvent for said indicator selected from the classconsisting of propylene Aglycol and ethylene glycol, said amount of saidsolvent being sufficient to maintain the solubility of said indicator insaid solution at a pH range of from about 4.0 to about 5.6, and anamount of a catalytic enzyme consisting of glucose oxidase buffered to apH in the range of from about 4.0 to about 5.6 to prevent said indicatorfrom being reoxidized suiciently to interfere with said color change ofsaid indicator at said predetermined glucose level, said amount of saidenzyme being suflicient to act as a catalyst in the reduction of saidindicator by said glucose, and warming said solution to a predeterminedtemperature for a period sutiicient to reduce said indicator.

l2. A self-indicating method for directly screening for glucose at apredetermined level in a biological fluid comprising the steps ofbringing together in solution under substantially anaerobic conditions apredetermined amount of glucose-containing biological fluid, an amountof an oxidation-reduction indicator selected from the class consistingof sodium 2,6-dichlorobenzenone-indophenol and sodium2,6-dibromobenzenone-indophenol, said amount of said indicator beingsufficient to change color as it is reduced in response to levels ofglucose in said biological fluid equal to and above said predeterminedlevel, and an amount of a catalytic enzyme consisting of glucose oxidasebuffered to a pH of from about 4.0 to about 5.6 to prevent saidindicator from being reoxidized su'ciently tov interfere with said colorchange of said indicator at said predetermined glucose level, saidamount of said enzyme being sufficient to act as a catalyst in thereduction of said indicator by said glucose, and warming said solutionto a predetermined temperature for a period suicient to reduce saidindicator.

References Cited in the tile of this patent UNITED STATES PATENTS2,893,844 Cook July 7, 1959 FOREIGN PATENTS 203,451 Australia Sept. 27,1956 OTHER REFERENCES Bacteriology and Allied Subjects, by Gershenfeld,1945, publ. by Mack Publishing Co., Pa., page 79.

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1. A SELF-INDICATING METHOD FOR DIRECTLY SCREENING FOR GLUCOSE AT APREDETERMINED LEVEL IN A BIOLOGICAL FLUID COMPRISING THE STEPS OFBRINGING TOGETHER IN SOLUTION A PREDETERMINED AMOUNT OFGLUCOSE-CONTAINING BIOLOGICAL FLUID, AN AMOUNT OF AN OXIDATION-REDUCTIONINDICATOR SELECTED FROM THE CLASS CONSISTING OF SODIUM2,6-DICHLOROBEZENONE-INDOPHENOL AND SODIUM2,6-DIBROMOBEZENONEINDOPHENOL, SAID AMOUNT OF SAID INDICATOR BEINGSUFFICIENT TO CHANGE COLOR AS IT IS REDUCED IN RESPONSE TO LEVELS OFGLUCOSE IN SAID BIOLOGICAL FLUID EQUAL TO AND ABOVE SAID PREDETERMINEDLEVEL, AND AN AMOUNT OF A CATALYTIC ENZYME CONSISTING OF GLUCOSE OXIDASEBUFFERED TO A PH